Photosynthesis
Quick Introduction
Photosynthesis is the metabolic process that provides usable energy for green plants by converting radiant energy from sunlight into chemical energy (in the forms of NADPH and ATP) and then using that chemical energy to reduce carbon dioxide to carbohydrates. The summary equation is:
carbon dioxide (CO2) + water (H2O) + light à glucose (C6H12O6) + oxygen (O2)
Note that in this process, carbon dioxide is reduced to glucose (with electrons and hydrogen from water) and water is oxidized to oxygen (by losing those electrons and hydrogen). The role of light absorption becomes clear when you consider that it is impossible (the reaction is non-spontaneous and endergonic) for carbon dioxide and water to react to produce glucose and oxygen without additional energy input. The process of light harvesting by plants is also very efficient: ATP and NADPH are produced at a sufficient rate to provide the energy to synthesize carbohydrates, and also create a surplus of ATP for use in other metabolic processes.
We will be studying photosynthesis in Azolla, a small aquatic fern. Azolla is a good experimental organism to investigate photosynthesis because it responds quickly to changes in light intensity and is small enough to easily fit in a reaction cuvette.
In a previous lab, respiration was studied by measuring the rate of CO2 formation using a sensor interfaced to a computer. Today, we will use the same apparatus except that the amount of CO2 in the cuvette should decrease with time as it is assimilated in photosynthesis.
What to do:
There are two RQ’s to be considered in today’s lab:
1) Is light required for photosynthesis?
2) What is the effect of light intensity on photosynthesis rate?
Pose a hypothesis from each RQ and design experiments to test your hypotheses. Answer the following questions: What are the controls? What are the experimental groups? Independent variable? Dependent variable? Remember to construct an appropriate data collection table in your lab notebook and check with the instructor before you proceed.
Tools:
CO2 probe set up
1. Make sure the CO2 probe is connected to the LabPro interface and the computer.
2. Make sure the tip of the probe is in air before you continue. Note: this probe will be ruined by immersion in liquid. Make sure that it never comes in contact with water.
3. On the right hand of the screen, click on the “Logger Pro” icon.
4. Check the boxed display. It should read approximately 400-500 ppm. If it is not, try to bring the reading down using the portable fan at your lab table. Remember that you exhale a lot of CO2. If you breathe directly on the sensor, it will be difficult to get the probe to an operating condition. If all else fails, your instructor can do a re-calibration of the probe.
5.
You are now ready to begin readings. In
subsequent experiments, make sure the starting CO2 level is the
same.
1. The high intensity light sources used in this exercise have the potential to overheat the reaction vessels and harm the ferns being studied in this lab. Infrared radiation is removed by placing a water filled vessel (a fish bowl) between the light source and the reaction cuvette. Periodically check the temperature of the water in the fish bowl; if it gets hot to the touch, replace it with cold water from the tap.
2. Remember to lineup the light source with the reaction cuvette so that the light uniformly illuminates the surface of the plants.
Setup for Measuring
Photosynthesis Rate
1. Place 50 ml of water in the reaction
cuvette (125 ml flask).
2. Determine the weight of the reaction
vessel and record the data in your notebook.
3. Using a tongue depressor, transfer enough
Azolla to the cuvette so that the surface of the water is covered with
one layer of plants.
4. Reweigh the reaction vessel and subtract
the weight in step 2 from this amount to determine the weight of the Azolla.
Record this value in your notebook.
5. Using the rubber stopper assembly, insert
the CO2 sensor in the flask.
6. Recheck and make sure that you have
positioned the flask in front of the light source so that the light uniformly
illuminates the surface of the plants.
7. Click
"collect" on the computer. It has been preset to collect CO2
data for 300 seconds (at 1 sample every 2 seconds). The computer will plot the
change in CO2 concentration per time on the screen as you watch.
8. Once the data gathering is complete, hold down the left hand button on the mouse and select the interval on the line representing a linear change in CO2 concentration. The interval should be for at least 60 seconds. The selected region should be highlighted. Select “analyze” on the menu across the top of the screen and then chose “linear fit”. A box should appear on the graph with the equation of the line and the correlation coefficient (goodness of fit for the data to the line). Record the slope in your data collection table. What are the units?
1. The rates as measured above reflect the rate of change of CO2 in the reaction vessel. In photosynthesis, as the amount of CO2 decreases with time, plants showing higher photosynthesis rates will have (paradoxically) greater negative slopes. This can be very confusing for beginning students, so by convention the sign of the slopes of the lines obtained from photosynthesis gas exchange experiments are reversed. This means that before you plot any of your photosynthesis data, first change the signs from negative to positive.
2. Also, remember to convert your raw data expressed as: photosynthesis rate (ppm CO2/s) into specific activity data (ppm CO2/s/g Azolla). You do this by dividing your raw rate data by the weight of Azolla recorded in your notebook.
How
to save data after a run:
1. Select "experiment" on the menu across the top of the screen and then chose "store latest run".
2. If you want to hide the data from a run, select “data” from the top menu and chose "hide data set". NOTE: Always choose “run 1” (or whatever number is appropriate) on the menu that appears. DO NOT CHOOSE “LATEST RUN”. If you do, on the next assay, this will cause the program to lock up and give the message, “waiting for data”. (If you make this mistake anyway, go to the “data” menu and select “show data set” and the problem will be resolved.)
1. The amount of light received by the reaction cuvette varies with the distance between the light source and the surface of the plants. We have previously determined this relationship; the data can be found here. Using this data, make a plot showing the relationship between light intensity (lux) and distance from the light source.
2. Choose 5 different distances to measure photosynthesis rate in Azolla. Remember to convert distance to light intensity before you plot your data.
Assignment
1. If time allows, sketch the final figure
from today’s results on the board
2. Create a computer generated figure of
your data using Excel:
http://homepage.mac.com/haleyub/Students/PhotoAlbum31.html
1. What was the relationship between light intensity and photosynthesis rates in your experiments? Was it linear? Nonlinear? Explain.
2. Did the results support your hypotheses?